Function of stimulus-induced MeCP2 phosphorylation Abstract MeCP2 is a key player in recognizing methylated DNA and interpreting the epigenetic information encoded in different DNA methylation patterns. Alterations in sequence or copy number of the X-linked human MECP2 gene cause either Rett syndrome (RTT) or MECP2 duplication syndrome. In addition to RTT-causing mutations, many missense mutations with unknown functional significance have been identified in the MECP2 gene in humans. Two of those mutations are at or close to serine 80 or serine 421, whose phosphorylation status is important for regulating MeCP2 function. To fully understand the significant role of MECP2 in regulating the development and function of the nervous system, it is important to study all aspects of MeCP2 function. We have previously demonstrated that phosphorylation at serine 421 (S421) can be induced by spatial learning, and that S421 phosphorylation plays critical roles in regulating MeCP2 binding to gene promoters, neuronal gene transcription, excitatory synaptogenesis, two types of synaptic plasticity (long-term potentiation and synaptic scaling), locomotion, learning and memory. Most recently, we discovered that S421 is also phosphorylated in adult neural progenitor cells (aNPC) isolated from the hippocampus. Interestingly, the stimulus, the regulation and the function of S421 phosphorylation in aNPCs are completely different from those in post-mitotic neurons. In aNPCs, MeCP2 S421 phosphorylation is induced by growth factors, linked to cell cycle, directly regulated by aurora kinase B, and plays critical roles in regulating the proliferation and differentiation of aNPCs through the Notch signaling pathway. These new findings further generalize MeCP2 phosphorylation as a common regulatory module in cellular functions. More interestingly, phosphorylation at serine 80 (S80) appears to be regulated differentially from phosphorylation at S421 in post-mitotic neurons, and plays opposing functional roles against S421 phosphorylation in both post-mitotic neurons and aNPCs. Collectively, these studies raise the possibility that dynamic phosphorylation states at multiple sites on MeCP2 may form a combinatorial code, presenting another epigenetic regulatory module in addition to DNA methylation and histone codes. To test this hypothesis, we propose to generate several novel Mecp2 knockin alleles carrying combinations of point mutations that either abolish or mimic phosphorylation at S80 and S421, and study the functional output of each combinatorial code in the well-established paradigm of neurogenesis in both mouse and human models. Our specific aims are: 1) To study the effects of double mutations at S80 and S421 on aNPC proliferation and differentiation in mouse models, 2) To study the effects of single and double mutations at S80 and S421 on NPC proliferation and differentiation in human stem cell models, and 3) To define the molecular mechanism linking MeCP2 phosphorylation with its functional output. Studying posttranslational modification of MeCP2 provides a critical angle of understanding the basic function of MeCP2, which will expand our knowledge of MeCP2 beyond RTT and MECP2 duplication syndrome.